Voltage-sensing domains (VSDs) are transmembrane protein modules that detect electrical signals propagating within cell membranes. Ion channels and enzymes containing these domains play key roles in many biological processes, from the generation of the action potential in neurons and muscles, to the regulation of reactive oxygen species (ROS) during infection and inflammation. Malfunction or misexpression of VSD-containing proteins is associated with numerous diseases, such as epilepsy, periodic paralysis, cardiac arrhythmia, cancer and autoimmune disorders. Some VSDs conduct ions across the membrane under physiological conditions. Others become ion permeant under pathological conditions, as a result of mutations. The long-term goal of this study is to elucidate the mechanism underlying ion conduction through the VSD and its relationship to the general mechanism of voltage sensing. The study focuses on the voltage-gated proton channel Hv1, a protein that lacks the pore domain typical of voltage-gated sodium, potassium, and calcium channels and conducts protons through its VSD. Recent work has begun to unveil the structural organization of Hv1, but many open questions remain about the mechanisms of proton permeation, gating, and modulation of the channel. In this study we plan to answer some of these questions by using an approach that combines electrophysiological and fluorescence techniques to mutagenesis scanning and molecular dynamics simulations. Specifically, we aim at: 1) determining which parts of the VSD make up the proton pore and gate by using a novel technique of perturbation analysis recently developed in our laboratory, 2) exploring the relationship between the mechanism of proton permeation through the VSD and the mechanism of voltage sensing, using new Hv1 blockers as molecular probes, and 3) investigating the mechanisms of subunit coupling and gating modulation by accessory proteins. The proposed research will significantly expand our understanding of how VSDs sense the membrane potential, conduct ions, and interact with intracellular processes via accessory proteins. The work will also pave the way to the development of Hv1 inhibitors that can be used to address ROS overproduction typical of several cardiovascular and inflammatory disorders and will provide new insights on how mutations of VSDs lead to disease.